I Discovery and Modulation of Acid-Sensing Ion
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Discovery and modulation of acid-sensing ion channel modulating venom peptides Ben Cristofori-Armstrong Bachelor of Science (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Faculty of Medicine, School of Biomedical Sciences I Abstract Acid-sensing ion channels (ASICs) are a family of proton-activated cation channels expressed in a variety of neuronal and non-neuronal tissues. As proton-gated channels, they have been implicated in many pathophysiological conditions where pH is perturbed. Venom derived compounds represent the most potent and selective modulators of ASICs described to date, and thus have been invaluable pharmacological tools to study ASIC structure, function, and biological roles. There are now eleven ASIC modulators described from animal venoms, with those from snakes and spiders favouring ASIC1, while the sea anemone and cone snail modulators preferentially target ASIC3. Chapter 1 reviews the current state of knowledge on venom derived ASIC modulators, with a particular focus on their molecular interaction with ASICs, what they have taught us about channel structure, and what they may still reveal about ASIC function and pathophysiological roles. Venom peptides are often disulfide bonded making their recombinant expression challenging. Chapter 2 describes a periplasmic Escherichia coli protocol for production of correctly folded peptides that was used throughout this thesis. Using the two-electrode voltage-clamp technique, Chapter 3 shows that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either Xenopus laevis or X. borealis oocytes. This voltage-clamp technique was heavily used throughout this thesis to perform functional studies of venom peptides. The spider venom peptide PcTx1 is the best studied ASIC modulator; it has an IC50 of ~1 nM at rat ASIC1a and is neuroprotective in rodent models of ischemic stroke. Chapter 4 examines the molecular interaction between PcTx1 and both human ASIC1a and the off- target ASIC1b subtype, where little experimental work has been done. We show that although PcTx1 is 10-fold less potent at human ASIC1a than the rat channel, the apparent affinity for the two channels is comparable. The pharmacophore of PcTx1 for human ASIC1a and rat ASIC1b was examined via alanine scanning mutagenesis and uncovered residues that show subtle ASIC1 species and subtype-dependent differences in activity that may allow for further manipulation to develop more selective PcTx1 analogues. The ASIC3 inhibitor APETx2 is analgesic in rodent models of chemically-induced, inflammatory and postoperative pain, providing strong evidence for the ASIC3 subtype as a potential pain target. Despite the comprehensive structure-activity studies of APETx2, the mechanism of action and channel binding site have remained elusive. Chapter 5 fills this II gap in knowledge of an important ASIC research tool and also reveals subtle differences in the pharmacophore of APETx2 for inhibition of ASIC3 and potentiation of ASIC1b. We propose that APETx2 binds to a novel region for peptide modulators of ASICs and acts by preventing the closed-to-open gating transition of rat ASIC3. Mambalgins are a group of three-finger toxins isolated from black and green mamba snake venoms that target ASIC1a and ASIC1b containing channels. Unique among ASIC modulators, the potent inhibitory activity of mambalgins at rodent ASIC1b was crucial in demonstrating that ASIC1b, and not ASIC1a, is important for peripheral pain sensing in rodents. Chapter 6 shows that the efficacy of mambalgins varies between the ASIC1 splice variants ASIC1a and ASIC1b, in both human and rat channels. Strikingly, mambalgin is a potentiator of human ASIC1b under certain conditions. Furthermore, these pharmacological differences are due to both the molecular interactions at the binding sites and the different ways mambalgin can modify the gating characteristics of specific ASIC variants. Chapters 7 describes the isolation, pharmacological characterisation and chemical stability of the novel spider venom peptide Hm3a from the venom of the tarantula Heteroscodra maculata. Hm3a is a close homolog of PcTx1 with five amino acid substitutions and a three residue C-terminal truncation. Despite its high sequence similarity with PcTx1 and similar pharmacology, Hm3a showed higher levels of stability over 48 h and will be particularly useful when stability in biological fluids is required, for example in long term in vitro cell- based assays and in vivo experiments. Chapter 8 describes the characterisation of conorfamides As1a and As2a from the venom of the cone snail Conus austini. Amidated peptide variants altered desensitization of ASIC1a and 3, and a lysine to arginine mutation introduced ASIC1a peak current potentiation. These conorfamides also inhibited a7 and muscle-type nicotinic acetylcholine receptors (nAChR) at nanomolar concentrations. These are the first conorfamides with the dual pharmacology described to date. III Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis. IV Publications included in this thesis 1. B Cristofori-Armstrong# and LD Rash# (2017) Acid-sensing ion channel (ASIC) structure and function: Insights from spider, snake and sea anemone. Neuropharmacology 127: 173–184. 2. SY Er*, B Cristofori-Armstrong*, P Escoubas and LD Rash (2017) Discovery and molecular interaction studies of a highly stable, tarantula peptide modulator of acid- sensing ion channel 1. Neuropharmacology 127: 185–195. 3. B Cristofori-Armstrong, MS Soh, S Talwar, DL Brown, JDO Griffin, Z Dekan, JL Stow, GF King, JW Lynch and LD Rash (2015) Xenopus borealis as an alternative source of oocytes for biophysical and pharmacological studies of neuronal ion channels. Scientific Reports 5: 14763. V Submitted manuscripts included in this thesis 1. B Cristofori-Armstrong*, NJ Saez*, IR Chassagnon, GF King, and LD Rash. The modulation of acid-sensing ion channel 1 by PcTx1 is pH-, subtype- and species- dependent: importance of interactions at the channel subunit interface and potential for engineering selective analogues. Biochemical Pharmacology. Revisions resubmitted. 2. A-H Jin*, B Cristofori-Armstrong*, LD Rash, SAR González, RJ Lewis, PF Alewood, and I Vetter. Novel conorfamides from Conus austini venom modulate both nicotinic acetylecholine receptors and acid-sensing ion channels. Biochemical Pharmacology. Submitted. Other publications during candidature Book Chapter 1. NJ Saez, B Cristofori-Armstrong, R Anangi, and GF King (2017) A Strategy for Production of Correctly Folded Disulfide-rich Peptides in the Periplasm of E. coli. Methods in Molecular Biology 1586: 155–180. Journal Article (* joint first author; # co-corresponding author) 1. A Silva*, B Cristofori-Armstrong*, LD Rash, WC Hodgson and GK Isbister (2018) Defining the role of post-synaptic -neurotoxins in paralysis due to envenoming in humans. Cellular and Molecular Life Sciences 75: 4465–4478 2. H Shen, Z Li, Y Jiang, X Pan, J Wu, B Cristofori-Armstrong, JJ Smith, YKY Chin, J Lei, Q Zhou, GF King, N Yan (2018) Structural basis for the modulation of voltage-gated sodium channels by animal toxins. Science 362: eaau2596 3. B Madio, S Peigneur, YKY Chin, BR Hamilton, ST Henriques, JJ Smith, B Cristofori- Armstrong, Z Dekan, BA Boughton, PF Alewood, J Tytgat, GF King and EAB Undheim (2018) PHAB toxins: a unique family of sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold. Cellular and Molecular Life Science 75: 4511–4524. 4. JYP Lee*, NJ Saez*, B Cristofori-Armstrong*, R Anangi, GF King, MT Smith and LD Rash (2018) Inhibition of acid-sensing ion channels by diminaene and APETx2 evoke VI partial and highly variable antihyperalgesia in a rat model of inflammatory pain. British Journal of Pharmacology 175: 2204–2218. 5. SS Pineda, PA Chaumeil, A Kunert, Q Kaas, MWC Thang, L Li, M Nuhm, V Herzig, NJ Saez, B Cristofori-Armstrong, R Anangi, S Senff, D Gorse and GF King (2017) ArachnoServer 3.0: an online resource for automated discovery, analysis and annotation of spider toxins. Bioinformatics 34: 1074–1076. 6. JS Wingerd, CA Mozar, CA Ussing, SS Murali, YKY Chin, B Cristofori-Armstrong, T Durek, J Gilchrist, CW Vaughan, F Bosman, DJ Adams, RJ Lewis, PF Alewood, M Mobli, MJ Christie and LD Rash (2017) The tarantula toxin /-TRTX-Pre1a highlights the importance of the S1-S2 voltage-sensor region for sodium channel subtype selectivity. Scientific Reports 7: 974.